Recognition studies using monoclonal antibodies. During the first year of this project we demonstrated antibody recognition of vesicle-associated antigen by the ability of lipid vesicles to inhibit antibody and complement-mediated lysis of murine target cells. Although this assay is highly reproducible and specific, we have attempted during this year to devise more sensitive assays for recognition by monoclonal antibodies. To measure lipid vesicle-bound antibody, we have developed a complement-depletion assay in which vesicles and antibody are incubated (with or without murine target cells) with limiting amounts of complement at 37~. Supernatants are then harvested and tested for residual complement against sensitized sheep erythrocytes. While we can demonstrate complement depletion by lipid vesicle-bound antibodies, to date this assay is comparable in sensitivity to measuring inhibition of lysis as mentioned above. We have attempted to measure direct binding of monoclonal antibodies to lipid vesicles using both iodinated and metabolically labeled (35S-methionine, 3H-leucine) antibodies. For such studies efficient separation of bound and unbound antibodies is critical. To achieve this we have separated lipid vesicles by centrifugation (including sucrose gradients and mixtures of oils of different densities), gel filtration and differential precipitation by polyethylene glycol. Presently, we are unable to get satisfactory separations using these procedures and are experimenting with polycarbonate filters and lectin-agarose immobilization assays. We have obtained three alloreactive (C57BL/6 anti-DBA/2) T-cell lines cloned by limiting dilution from bulk mixed lymphocyte culture (MLC) and maintained in the presence of irradiated stimulator cells and secondary MLC supernatant as a source of T-cell growth factors (TCGF). These clones are tested regularly for their ability to proliferate in response to antigen in the presence and in the absence of added growth factors and for their cytolytic activity. Macrophages from mice infected with Mycobacterium bovis, strain BCG, effectively and selectively bind tumor cells in vitro, a necessary but not sufficient step in target cell cytolysis. The process of augmented binding, starting with the initial contact of the two cells and proceeding to a firm, rigorous interaction, is currently under study.